Inherent properties of a naturally occurring or "wild type"0 enzyme are not necessarily optimized for catalytic utilization of the enzyme outside of the native biological environment. Use of the isolated enzyme in non-native non-biological environments is often limited by such properties as the enzyme's substrate specificity, thermostability, activity levels under various conditions (e.g., temperature and pH), oxidation stability, and the like. It may thus be desirable to alter a natural property of an enzyme to optimize a certain characteristic of the enzyme for a specific use, or for use in a specific non-native non-biological environment (Klibanov, A.M., Chemtech 16:354 (1986); Zaks, A. et al., J. Biol. Chem. 263:3194 (1988)).
Enzymatic catalysts have been much desired in the area of organic syntheses, especially, acylation reactions, for example, in the in vitro chemical synthesis of nucleosides and peptides, and in the resolution of chiral alcohols and acids, and the incorporation of D-amino acids into peptides. However, enzymatic catalysts are not generally useful in organic syntheses because of the catalytic instability of natural enzymes in many of the relatively harsh reaction environments routinely used to drive the syntheses reactions, such as high concentrations or organic solvents and/or high temperatures.
One area where an enzymatic catalyst is greatly desired is in those organic syntheses which involve selective acylation reactions such as those which occur in the regioselective acylation of sugars and nucleosides. Selective chemical acylations of nucleosides have been reported in only a few cases (Brown, D. M. et al., J. Chem. Soc. K. et al., Nucleic Acids Res. Symposium Series No. 16, IRL Press, Oxford, pp. 177-180 (1985); Liguori, A. et al., Tetrahedron 44:229-234 (1988); Mitsunobu, O. et al., Chem. Soc. Jpn. 45:245 (1972); Shimokawa, S. et al., Chem. Soc. Jpn. 49:3357-3358 (1976)). Previously, to selectively acetylate the more reactive 2'-hydroxyl positions, the amount of acylating agent employed was limited (Kamaike, K. et al. (Nucleic Acids Res. Symposium Series No. 16, IRL Press, Oxford, pp. 177-180 (1985)). Liguori, et al. (Tetrahedron 44:229-234 (1988)) recently reported the selective acylation of certain 2'-deoxynucleosides by use of a highly hindered mixed anhydride. Regioselective formation of the 5'-0-acyl groups have been accomplished by in situ displacement of an activated 5'-hydroxyl derivative (Mitsunobu, O. et al., Chem. Soc. Jpn. 45:245 (1972); Shimokawa, S. et al., Ibid 49:3357-3358 (1976)). This last procedure allows distinct time savings to be achieved over the traditional three step approach (Brown, D. M. et al., J. Chem. Soc. 3299 (1950)) but is not an acylation reaction in the formal sense.
Other methods involving selective deacylation of fully blocked nucleosides have also been used to prepare the 5'-0-acylnucleosides (Ihido, Y. et al., J. chem. Soc. (Perkin Trans. I) 2088 (1979)). Regioselective enzymatic acylations of certain nucleosides have recently been accomplished using subtilisin Carlsberg (Riva, S. et al., J. Am. Chem. Soc. 110:584-589 (1988)) with moderate to good efficiency. The utility of a recently reported efficient deoxygenation (Prisbe, E. J. et al., Synth. Commun. 15:401-409 (1985)) leading to synthesis of the AIDS virus (HIV) inhibiting 2',3'-dideoxynucleosides (Mitsuya, H. et al., Proc. Nat'l. Acad. Sci. USA 83:1911 (1986)) suffered from the losses associated with preparing the starting materials, the 5'-0-acetyl-2-deoxynucleosides. For example, 5'-0-acetylthymidine was obtained from thymidine in approximately 50% yield over two steps (Ihido, Y. et al., J. Chem. Soc. (Perkin Trans. I) 2088 (1979). Methods for the efficient synthesis of the 2',3' -dideoxynucleosides are highly desirable as 2',3'-dideoxynucleosides, especially 2',3'-dideoxyadenosine and 2',3'-dideoxyinosine, have utility as antiviral agents, including anti-HIV activity (Mitsuya, H. et al., Proc. Natl. Acad. Sci. USA 83:1911-1915 (1986), and as reagents for DNA sequencing (Sanger, F. et al., Proc. Natl. Acad. Sci. USA 74:5463 (1977). Existing methods for the preparation of these nucleosides are tedious and result in a low yield (Pfitzner, K. E. et al., J. Org. Chem. 29:1508 (1964); McCarthy, J. R. et al., J. Am. Chem. Soc. 88:1549 (1966); Michelson, A. M., et al., J. Chem. Soc., 816 (1955); Horwitz, J. P., et al., J. Org. Chem. 32:817 (1967); and Samukov, V. V. et al., Bioorg. Khim. 9:132 (1983)). Thus a need exists for a method of synthesizing precursors to biomedically important nucleosides in an efficient and specific manner.
Another area where an enzymatic catalyst is greatly desired is in peptide synthesis. The possibility of using proteases to catalyze the formation of a peptide bond rather than the hydrolysis and cleavage of a peptide bond was first discussed by van't Hoff, J. H. et al., Chem. 18:1 (1898), and experimentally demonstrated by Bergmann, M. et al., J. Biol. Chem. 124:1 (1938). However, although the idea of using proteases in organic peptide syntheses as peptide ligases has been widely examined and desired (Margolin, A. L. et al., J. Am. Chem. Soc., 109:7885-7887 (1987); Fruton, J.S., Adv. Enzymol. 53:239-306 (1982); Chaiken, I. M. et al., Appl. Biochem. Biotechnol. 7:385-399 (1982); Jakubke, H.-D. et al., Angew. Chem. Int. Ed. Engl. 24:85-93 (1985); and Kullman, W., Enzymatic Peptide Synthesis, CRC Press, Boca Raton, Flor. (1987)), the application of proteases to peptide syntheses has been limited by the lack of a protease which is catalytically stable in the reaction environments required for in vitro amino acid or peptide ligation and synthesis.
A subtilisin is a serine protease naturally produced by Gram positive bacteria and by fungi. A serine protease is an endoprotease which catalyzes the hydrolysis of a peptide bond in which there is an essential serine residue at the active site. Serine proteases can be inhibited by phenylmethanesulfonylfluoride and by diisopropyl fluorophosphate.
The use of native bacterial subtilisin to catalyze peptide bond formation in the presence of a water-miscible solvent is known (Barbas, C.F. et al., J. Am. Chem. Soc. 110:5162-5166 (1988); West, J. B. et al., J. Am. Chem. Soc. 110:3709 (1988); Margolin, A. L. et al., J. Am. Chem. Soc. 109:7885-7887 (1987); and Gross, A. T., European Patent Application Publication No. 272,564.) Bacterial subtilisin has been reported to be active in a number of anhydrous organic solvents, including tert-amyl alcohol, tetrahydrofuran (THF), acetone, acetonitrile, ethyl acetate and dioxane. It is also known that subtilisin catalyzes the formation of a peptide bond between an N-protected amino acid chloroethyl ester and an amino acid amide. However, the native subtilisin enzyme is unable to catalyze peptide bond formation when it is used in an environment comprising an aqueous solution containing 40% N,N-dimethylformamide (DMF) at either pH 7 or pH 10, an environment often desired in organic syntheses.
Thiolsubtilisin is a chemically altered derivative of native subtilisin (Neet, K.E. et al., Proc. Nat'l Acad. Sci. USA 56:1606 (1966), and Philipp, M. et al., Mol. Cell Biochem. 51:5 (1983)). Thiolsubtilisin is a very poor protease but retains the ability to catalyze the reverse reaction, that of peptide ligation, and hence is able to catalyze the formation of a peptide bond. The use of thiolsubtilisin as a catalyst for peptide condensation has been disclosed (Nakatsuka, T. et al., J. Am. Chem. Soc. 109:3808-3810 (1987)). Thiolsubtilisin was found to be too inactive or too slow in anhydrous DMF to be used for preparative peptide synthesis. However a high-yield of peptide bond formation was achieved in aqueous DMF (50% water v/v).
The subtilisin gene from Bacillus amyloliquefaciens (subtilisin BPN') has been cloned and expressed at high levels from its natural promoter sequences in Bacillus subtilis (Vasantha, N. et al., J. Bact. 159:811 (1984); Wells, J. A. et al., Nucleic Acid Res. 11:7911 (1983)). This has enabled the introduction of mutations in vitro into the plasmid-encoded subtilisin gene and allowed the analysis of the effect of those mutations on the properties of the altered enzyme. Mutant subtilisin genes have been cloned into a strain of B. subtilis which contains a chromosomal deletion of its subtilisin gene and therefore produces no background wild type activity. Most mutant enzymes are efficiently expressed from this vector and are secreted into the culture medium at a concentration of about 1 g/L. Subtilisin is the major secreted protein in this system and comprises almost 80% of the total extracellular protein (Bryan, P. N. et al., Proc. Natl. Acad. Sci. USA 83:3743 (1986)).
The amino acid sequences of at least six subtilisins are known. These include five subtilisins from Bacillus strains (subtilisin BPN', subtilisin Carlsberg, subtilisin DY, subtilisin amylosacchariticus, and mesenticopeptidase) (Vasantha, N. et al., J. Bacteriol. 159:811-819 (1984); Jacobs et al., Nucleic Acids Res. 13:8913-8926 (1985); Nedkov et al., Biol. Chem. Hoppe-Seyler 366:421-430 (1985); Kurihara et al., J. Biol. Chem. 247:5619-5631 (1972); and Svendsen et al., FEBS Lett. 196:228-232 (1986) and the subtilisin thermitase from Thermoactinomyces vulgaris (Meloun et al., FEBS Lett. 183:195-200 (1985)).
The amino acid sequences from two fungal proteases which are closely related to subtilisin BPN' are also known: proteinase K from Tritirachium album (Jany et al., Biol. Chem. Hoppe-Seyler 366:485-492 (1985)) and thermomycolase from the thermophilic fungus, Malbranchea pulchella (Gaucher et al., Methods Enzymol. 45:415-433 (1976)). These enzymes have been shown to be related to subtilisin BPN', not only through their primary sequences and enzymological properties, but also by comparison of x-ray crystallographic data. (McPhalen et al., FEBS Lett. 188:55-58 (1985); and Pahler et al., EMBO J. 3:1311-1314 (1984)).
Through the techniques of site specific mutagenesis nearly any amino acid position in a target protein can be manipulated at will (Smith, M., Ann. Rev. Genet. 19:423 (1985)). Much of the past efforts in this field have been directed at elucidation and manipulation of the active site regions of various enzymes (Gerlt, J. A., Chem. Rev. 87:1079 (1987)). Site specific mutagenesis has served as a means of creating proteins with improved characteristics for specific industrial and therapeutic uses (Bryan, P. N., Biotechnology Advances 5:221 (1987)). Reported modifications of the subtilisin gene have been directed to increasing the thermostability of the enzyme (Bryan, P.N. et al., Proteins: Structure, and Genetics 1:326; (1986); Pantoliano, M. W. et al., Biochemistry 26:2077 (1987); Estell, D. A. et al., J. Biol. Chem. 260:6518 (1985); Bryan, P. et al., Proc. Nat'l. Acad. Sci. USA 83:3743 (1986); Wells, J. A. et al., Philos. Trans. R. Soc. London A 317:415 (1986); Estell, D. A. et al., Science 233:659 (1986); Well, J. A. et al., Proc. Nat'l Acad. Sci. USA 84:1219 (1987)). An enzyme more stable to oxidative environments was produced by replacement of an oxidatively Tabile methionine residue (Estell, D. A. et al., J. Biol. Chem. 260:6518 (1985)). The inclusion of a disulfide bridge between residues 22 and 87 contributed approximately 1.3 kcal/mol to the free energy of unfolding (Pantoliano, M. W. et al., Biochemistry 26:2077 (1987)). This is significant since the total energy of unfolding for most proteins is only 5-15 kcal/mol (Creighton, T. E., Proteins: Structure and Molecular Properties W. H. Freeman, New York (1984)). Improvement of certain hydrogen-bonding interactions by a single point mutation increased the half-life of thermal inactivation four-fold over the native enzyme (Bryan, P. N. et al., Proteins: Structure, and Genetics 1:326 (1986)).
However, none of the prior art recognized that subtilisins which have been mutated to be more stable at higher temperatures may also possess new, unobvious advantages which make them highly useful in organic syntheses in the presence of organic solvents. Thus there remains a need for a catalytically stable enzyme catalyst capable of catalyzing chemical reactions in non-native organic environments in an efficient manner suitable for the bulk production of the product.